Recent advances in analytical methods for identification and quantification of phenolic compounds

Phenolic compounds are one of the largest groups of secondary metabolites present in the Plant Kingdom. This class of natural products has a wide range of biological activity, ranging from beneficial to adverse, in both humans and animals. Recent advances made in the analysis of simple phenolics and polyphenolics (tannins) are reviewed. For simple phenolic compound mixtures, choice of a separation and identification method is dependent on factors such as the physico-chemical properties (polarity, thermal stability and molecular weight) and the number of components in the mixtures. Gas chromatography is the method of choice for low molecular weight aromatic acids, alcohols and simple flavonoid aglycones. More polar and thermally unstable compounds, mainly glycosidic conjugates of phenolic acids and flavonoids, are separated well on reversed phase liquid chromatography (LC) columns. The most flexible separation method is capillary electrophoresis, where the highest sensitivity and separation can be achieved. Many different detectors [ultra violet (UV), mass spectrometry (MS), nuclear magnetic resonance (NMR), electrochemical, laser induced fluorescence (LIF)] are used for the identification of compounds eluted from the chromatographic columns. The UV and MS detectors are used widely due to their efficient provision of information, their high sensitivity and ease of use. Tannin analysis remains highly problematic. Reversed-phase high-pressure liquid chromatography (HPLC) has been used for separation of low molecular weight polymers. However, this technique has not been particularly useful for complex hydrolysable tannins (HT) and condensed tannin (CT) polymers beyond tetramers. The UV detection method is widely used because of its simplicity. However, it is not specific due to interference by other moieties present in plant extracts. Fluorescence and electrochemical detection systems can also be used. Structural information can be generated using MS and NMR approaches. Most tannin-containing plants contain both HT and CT, but unfortunately usually only CT (proanthocyanidins), are analysed. Furthermore, the biological effects are ascribed mostly to the CT, which might be misleading. There are two new assays for HT. These are based on their hydrolysis to gallic acid and then its determination, using HPLC. The second is based on the methanolysis of HT to methyl gallate and then its determination using potassium iodate; protein precipitation assays, both isotopic (based on I-125-labelled bovine serum albumin) and non-isotopic, representing the operational property of both HT and CT. There is a tannin bioassay based on an in vitro simulation of the rumen and measurement of tannin activity for both free and bound tannins in terms of rumen fermentation parameters; a C-14- polyethylene glycol binding assay; and a C-13-NMR-based assay will also be presented.